Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.26 (GSK)
 6,788 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Four distinct tyrosine protein kinases active on poly(Glu4,Tyr1) and angiotensin II, and operationally termed TPK-I, TPK-IIA, TPK-IIB and TPK-III have been resolved and partially purified from rat spleen particulate fraction by combining DEAE-Sepharose, heparin-Sepharose, phosphocellulose and polylysine-agarose chromatographies. Once partially purified all of them are free of Ser/Thr-specific protein kinase activity as judged using casein, histones, protamine and the peptide Arg-Arg-Ala-Ser-Val-Ala as substrates. TPK-I (apparent molecular mass 64 kDa, by gel filtration) and TPK-IIA (54 kDa) share several properties, including substrate specificity and stimulation by heparin; the latter however is much more responsive to polylysine then the former (10- and 3-fold maximum stimulation, respectively). Conversely TPK-IIB (51 kDa) is markedly inhibited by heparin and it is also characterized by its unique substrate specificity: unlike the other three tyrosine protein kinases it by far prefers the tetrapeptide Glu-Tyr-Ala-Ala over the decapeptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly and readily phosphorylates band-3 protein of red cell membrane. The unusual preference for Mg2+ over Mn2+ as activator and the capability to phosphorylate calmodulin distinguish TPK-III (61 kDa) from the other isoenzymes. Moreover TPK-III is insensitive to heparin and polylysine and is inhibited by quercetin much more efficiently than the other enzymes (I50 = 10 microM). Upon incubation with [gamma-32P]ATP, TPK-I, TPK-IIA and TPK-III give rise to alkali-stable radiolabeled components of 61, 55 and 52 kDa respectively, as evaluated by PAGE/SDS. In every case such a radiolabeling takes place also in the presence of a large excess of phosphorylatable substrate (angiotensin II) while it is readily reversed by isotopic dilution with 10-fold excess unlabeled ATP, supporting the view that it represents an autophosphorylation process. No (auto)phosphorylation product(s) could be detected in TPK-IIB even if its amount, in terms of catalytic activity, was 10-fold higher than that of the others.
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PMID:Characterization of four tyrosine protein kinases from the particulate fraction of rat spleen. 335 7

Tyrosine protein kinase activities were detected in the cytosolic fraction (PC-TPK) and the particulate fraction (PM-TPK) in human platelets using the synthetic peptide, E11G1 (Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly) as a substrate. PC-TPK and PM-TPK were different in substrate specificities, divalent cation requirements and apparent Mr values. These results strongly suggest that in platelets there exist at least two separate tyrosine protein kinases; one is present in cytosol and the other might be associated with membranes.
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PMID:Two separate tyrosine protein kinases in human platelets. 403 73

Inhibitor-2 (I-2) inhibits the free catalytic subunit of type 1 phosphatase (CS1) and controls the cyclic inactivation/activation of CS1 in the ATP-Mg-dependent protein phosphatase complex. We report here the effect of mutations on these two properties of I-2. Substitution of Thr-72 with Ala, Asp, or Glu generated complexes with CS1 that could not be activated. Mutation of Ser-86 did not affect activation by glycogen synthase kinase-3 (GSK-3) alone but impaired synergistic activation by casein kinase II and GSK-3. Mutations in the region between Thr-72 and Ser-86 did not alter the inhibitory potency of I-2 but prevented complete inactivation of CS1. A mutant without the 35 NH2-terminal residues exhibited an IC50 for CS1 200-fold higher than that of wild-type I-2. However, it formed an inactive phosphatase complex with CS1, which was activated by GSK-3. A mutant with the 59 COOH-terminal residues deleted retained full inhibitory activity and formed an inactive complex that could not be activated by GSK-3. We conclude that the NH2-terminal region of I-2 is involved in inhibition, that the sequence between Thr-72 and Ser-86 is necessary for the conversion of CS1 from an active to an inactive conformation, and that the COOH terminus is required for activation by GSK-3. Thus, different functional domains of I-2 may interact with distinct regions of CS1.
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PMID:Domains of phosphatase inhibitor-2 involved in the control of the ATP-Mg-dependent protein phosphatase. 796 54

Inhibitor-2 (I-2) is the regulatory subunit of the ATP-Mg-dependent phosphatase, a cytosolic form of type 1 protein phosphatase. Phosphorylation of I-2 at Thr-72 by the protein kinase glycogen synthase kinase-3 (GSK-3) leads to activation of the enzyme. Casein kinase II action was shown to synergistically enhance phosphorylation and activation by GSK-3 (DePaoli-Roach, A.A. (1984) J. Biol. Chem. 259, 12144-12152). Rabbit skeletal muscle and liver I-2 cDNA clones have been isolated. Rabbit skeletal muscle cDNAs could be placed in two subtypes, differing in the length of the 3'-untranslated region. The coding sequence of 612 nucleotides was identical in the two skeletal muscle and the liver cDNAs and predicted a protein of 204 amino acids, consistent with analysis of the purified protein. Northern hybridization analysis indicated that the two mRNAs of 1.7 and 2.7 kilobase pairs were present in all rabbit tissues examined, except in liver, where only the larger transcript was detected, and in testis, where additional transcripts were present. Expression in Escherichia coli of wild-type and phosphorylation site mutants resulted in the production of I-2 polypeptides with apparent M(r) values of approximately 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitory activity of the recombinant proteins was similar to that of native rabbit skeletal muscle I-2 and was unaffected by the substitution of alanine for the GSK-3 site (Thr-72) and for the casein kinase II sites (Ser-86 and Ser-120/121) or by substitution of glutamic acid and aspartic acid for Thr-72 and Ser-86. Recombinant wild-type I-2 and the Ala-120/121 mutant were phosphorylated synergistically by GSK-3 and casein kinase II. The Thr-72 and Ser-86 mutants, however, did not undergo this synergistic phosphorylation. Our studies indicate that Thr-72 is the only GSK-3 site and that Ser-86 is the casein kinase II site required for the potentiation of GSK-3 action. Furthermore, acidic residues cannot substitute for the phosphate group either in enhancing GSK-3 phosphorylation or in activating the phosphatase.
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PMID:Molecular mechanism of the synergistic phosphorylation of phosphatase inhibitor-2. Cloning, expression, and site-directed mutagenesis of inhibitor-2. 828 48

The transforming gene of Abelson murine leukaemia virus (v-abl) codes for a membrane-associated tyrosine-specific protein kinase (abl TPK). Analysis of the v-abl gene has shown that both the fibroblast-transforming and tyrosine-protein kinase activities reside within a minimal region encoding a protein of 43 kDa (p43v-abl), which represents the most active, isolated form of this enzyme. Since the cellular substrates for p43v-abl are yet to be identified, we synthesized by classical solution methods the octapeptide H-Gly-Asp-Thr-Tyr-Thr-Ala-His-Ala-OH, corresponding to the structural sequence of the main putative autophosphorylation site (Tyr 515) of the abl TPK, as well as some of its analogs modified in positions -2, -1, +1 and +3. The synthetic peptides were tested as substrates for the p43v-abl. The kinetic data obtained indicate that the rates of their phosphorylation vary considerably depending on the sequence of the peptide, as expected. As a rule, no significant increment of the efficiency results from each substitution in the parent sequence. While the replacement of the two charged residues, namely Asp-2 and His-7, with neutral Ala is well tolerated, the substitution with amino acids bearing opposite charges is detrimental. The correlation between secondary structure of our synthetic octapeptides and their substrate recognition by p43v-abl was studied using CD and fluorescence spectroscopy in 5 mM Tris, in 98% TFE/Tris and in 30 mM SDS solutions. The comparison of the spectroscopic data with the kinetic parameters does not confirm a close relationship between the conformational properties of these peptides and their enzymatic role.
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PMID:Synthesis and conformational studies on peptides corresponding to a putative autophosphorylation site of abl TPK. 846 52

A systematic analysis reveals that out of 20 protein kinases examined, specific for either Ser/Thr or Tyr, the majority are extremely sensitive to staurosporine, with IC50 values in the low nanomolar range. A few of them however, notably protein kinases CK1 and CK2, mitogen-activated protein (MAP) kinase and protein-tyrosine kinase CSK, are relatively refractory to staurosporine inhibition, exhibiting IC50 values in the micromolar range. With all protein kinases tested, namely PKA, CK1, CK2, MAP kinase (ERK-1), c-Fgr, Lyn, CSK and TPK-IIB/p38Syk, staurosporine inhibition was competitive with respect to ATP, regardless of its inhibitory power. In contrast, either uncompetitive or noncompetitive kinetics of inhibition with respect to the phosphoacceptor substrate were exhibited by Ser/Thr and Tyr-specific protein kinases, respectively, consistent with a different mechanism of catalysis by these two sub-families of kinases. Computer modeling based on PKA crystal structure in conjunction with sequence analysis suggest that the low sensitivity to staurosporine of CK2 may be accounted for by the bulky nature of three residues, Val66, Phe113 and Ile174 which are homologous to PKA Ala70, Met120 and Thr183, respectively. In contrast these PKA residues are either conserved or replaced by smaller ones in protein kinases highly sensitive to staurosporine inhibition. On the other hand, His160 which is homologous to PKA Glu170, appears to be responsible for the unique behaviour of CK2 with respect to a staurosporine derivative (CGP44171A) bearing a negatively charged benzoyl substituent: while CGP44171A is 10- 100-fold less effective than staurosporine against PKA and most of the other protein kinases tested, it is actually more effective than staurosporine for CK2 inhibition, but it looses part of its efficacy if it is tested on a CK2 mutant (H160D) in which His160 has been replaced by Asp. It can be concluded from these data that the catalytic sites of protein kinases are divergent enough as to allow a competitive inhibitor like staurosporine to be fairly selective, a feature that can be enhanced by suitable modifications designed based on the structure of the catalytic site of the kinase.
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PMID:Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2. 852 58

Eukaryotic initiation factor eIF2B is a guanine nucleotide exchange protein involved in regulation of translation initiation. Phosphorylation of the epsilon-subunit is thought to be important in insulin-mediated changes in eIF2B activity. However, elucidation of insulin's action has proven elusive, primarily because eIF2B epsilon is a substrate in vitro for at least three different protein kinases. In the present study, we observed changes in eIF2B epsilon kinase activity only in those muscles previously shown to exhibit alterations in protein synthesis in response to insulin. Specifically, eIF2B epsilon kinase activity was increased in psoas muscle from diabetic rats compared to controls. Treating diabetic rats with insulin rapidly reduced eIF2B epsilon kinase activity below control values. Changes were not observed in heart. To identify the kinase(s) in psoas responsible for phosphorylating eIF2B epsilon, the wildtype and two variant forms of the epsilon-subunit were expressed in and purified from Sf9 insect cells, and were used as substrates in protein kinase assays. The first variant contained a point mutation in the eIF2B epsilon cDNA that converted the glycogen synthase kinase-3 (GSK-3) phosphorylation site, Ser535, to a nonphosphorylatable Ala residue. In the second variant, the putative GSK-3 'priming' site, Ser539, was converted to Asp. Based on the pattern of phosphorylation of the wildtype and two variant forms of eIF2B epsilon using casein kinase (CK)-I, CK-II, or GSK-3 as well as that observed with skeletal muscle extracts, we conclude that the predominant eIF2B epsilon kinase in psoas muscle is GSK-3. Thus, insulin-mediated changes in eIF2B activity are likely to involve GSK-3.
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PMID:Glycogen synthase kinase-3 is the predominant insulin-regulated eukaryotic initiation factor 2B kinase in skeletal muscle. 1021 53

Glycogen synthase kinase-3 is involved in diverse functions including insulin signalling and development. In a number of substrates, phosphorylation by glycogen synthase kinase-3 is known to require prior phosphorylation at a Ser in the +4 position relative to its own phosphorylation site. Here we have used synthetic peptides derived from a putative glycogen synthase kinase-3 site in the Drosophila translation initiation factor eIF2B epsilon to investigate the efficacy of residues other than Ser(P) as priming residues for glycogen synthase kinase-3beta and its Drosophila homologue Shaggy. Glycogen synthase kinase-3beta phosphorylated peptides with Ser(P) and Thr(P) in the priming position, but peptides with Tyr(P), Thr, Glu or Asp were not phosphorylated. The Vmax for the Thr(P) peptide was three times higher than that of the Ser(P) peptide. These data suggest that glycogen synthase kinase-3 is unique among phosphate-directed kinases. The priming site specificity of Shaggy is similar to that of mammalian glycogen synthase kinase-3beta. This unpredicted efficacy of Thr(P) in the priming position suggests that there may be other unidentified substrates for these kinases.
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PMID:Phosphorylated seryl and threonyl, but not tyrosyl, residues are efficient specificity determinants for GSK-3beta and Shaggy. 1021 15

We have previously shown that PTH induction of c-fos expression in the rat osteoblastic cell line UMR 106-01 requires the phosphorylation of cAMP response element-binding protein (CREB) at serine 133. Here we show that this event is not sufficient for induced transcriptional activity in UMR cells. Serine 129, but not the casein kinase II sites (serines 108, 111, 114, 117, and 121), also plays a role in the activation of CREB. First, by metabolically labeling an epitope-tagged CREB, we determined that, in addition to serine 133, other residues are phosphorylated in vivo. Using mutational analysis of a GAL4-CREB reporter system we demonstrate that serines 129 and 133 are both required for PTH-induced transcriptional activity, whereas the casein kinase II sites are not. Furthermore, PTH failed to induce transcriptional activity of GAL4-CREB in cells treated with genistein, a general tyrosine kinase inhibitor known to inhibit glycogen synthase kinase-3 (GSK-3) activity, or LiCl, the most specific GSK-3-inhibiting agent known, strongly implicating GSK-3beta in this process. Importantly, although genistein and LiCl each inhibit GSK-3beta activity, neither prevented the phosphorylation of serine 133 induced by PTH. Lastly, when serine 129 is replaced with a negatively charged aspartic acid, LiCl has no effect on the PTH-induced trans-activation of CREB. We propose that GSK-3beta phosphorylates CREB at serine 129 and thus is required for the increased transcriptional activity of CREB in response to PTH.
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PMID:PTH induction of transcriptional activity of the cAMP response element-binding protein requires the serine 129 site and glycogen synthase kinase-3 activity, but not casein kinase II sites. 1179 24

Lithium has been used as an effective mood-stabilizing drug for the treatment of manic episodes and depression for 50 years. More recently, lithium has been found to protect neurons from death induced by a wide array of neurotoxic insults. However, the molecular basis for the prophylactic effects of lithium have remained obscure. A target of lithium, glycogen synthase kinase 3 (GSK-3), is implicated in neuronal death after trophic deprivation. The mechanism whereby GSK-3 exerts its neurotoxic effects is also unknown. Here we show that lithium blocks the canonical c-Jun apoptotic pathway in cerebellar granule neurons deprived of trophic support. This effect is mimicked by the structurally independent inhibitors of GSK-3, FRAT1, and indirubin. Like lithium, these prevent the stress induced c-Jun protein increase and subsequent apoptosis. These events are downstream of c-Jun transactivation, since GSK-3 inhibitors block neuronal death induced by constitutively active c-Jun (Ser/Thr-->Asp) and FRAT1 expression inhibits AP1 reporter activity. Consistent with this, AP1-dependent expression of proapoptotic Bim requires GSK-3-like activity. These data suggest that a GSK-3-like kinase acts in tandem with c-Jun N-terminal kinase to coordinate the full execution of the c-Jun stress response and neuronal death in response to trophic deprivation.
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PMID:Lithium blocks the c-Jun stress response and protects neurons via its action on glycogen synthase kinase 3. 1291 27


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